β-Glucans are a family of polysaccharides that is heterogeneous with respect to size, solubility and molecular structure. β-Glucans are polymers of glucose and the linkages can be 1,3, 1,4 and 1,6. The glucose chains in the β-glucan polymer can be linear or branched, with one type of linkages (e.g. 1,3) or a mixture of two or more (e.g. 1,3-1,4 or 1,3-1,6). The structure of the β-glucan highly influences the physical and chemical properties.
The most well-known polysaccharides made up of glucose are starch and cellulose. Starch is composed of amylose which is a linear polysaccharide with α-1,4 linkages and amylopectin which is a polysaccharide with α-1,4 linkages and branches having α-1,6 linkages. Cellulose is a linear β-glucan with β-1,4 linkages only.
Other well-known polysaccharides are the insoluble β-1,3 glucans pachyman and curdlan. Pachyman is a β-1,3-glucan derived from the sclerotia of Poria cocos (a Basidiomycetes sp.) and curdlan is produced by Alcaligenes faecalis var. myxogenes10C3K. Laminarin is a storage glucan found in brown algae and is a linear polysaccharide made up of a 1,3-glucan with 1,6 linkages. The ratio 1,3 to 1,6 is 3:1 (Wikipedia-wikipedia.org/wiki/Laminarin).
The β-glucans oat and barley are the two main cereals in terms of industrial supply of β-glucans. Oat β-glucan is water soluble and extensively studied for its health effect. The fibre is built of β-1,3 and β-1,4 linkages. Barley glucan contains the same linkages and is also water soluble.
The structure of yeast β-glucan is different from the cereal β-glucan and that results in different characteristics. Instead of chains of 1,3 and 1,4 linked glucose units, yeast β-glucan is composed of 1,3 and 1,6 linked monomers. The yeast glucan is found primarily in the yeast cell wall. The yeast cell wall contributes to 15-30% of the total dry weight of the cell.
The main components of the yeast cell wall are polysaccharides, protein and some chitin. The polysaccharides can be divided in β-glucans and mannans, composed of mannose monomers. The build-up of the cell wall is organized in layers. Roughly, one can divide the wall in three layers (Klis, F. M., Cell Wall Assembly in Yeast, Yeast 10, 851-869, 1994). The first layer is the surface of the cell and is composed mainly of mannoprotein. Below the first layer there is an inner layer between the surface and the plasma membrane and which consists of two layers. The inner layer closest to the plasma membrane is a fibrillar layer formed by β-1,3-glucan and the outer layer linking the inner layer and the surface is a more amorphous layer and is enriched in β-1,6-glucan chains to form a network. These components are cross-linked in various ways to form higher-order complexes. Covalent linkages are present between each of these components. The β-1,3-glucan is organized in helices, formed of either 1 or 3 chains of β-glucan. There are only a few branching points (β-1,6-linkage) in the network. The helices further reduce the solubility of the poorly water-soluble glucan. A small amount of chitin is also present which contributes to the insolubility of the fibres.
For the degradation of the various polysaccharides that occur in nature, many different enzymes have been identified. These so-called glycosidases or glycoside hydrolases (EC 3.2.1.x) form a large group of enzymes catalysing the hydrolysis of glycosidic bonds in oligo and polysaccharides. On cazy.org a continuously updated database can be found with all carbohydrate-active enzymes which are now categorized into 131 glycoside hydrolase families-see Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res 37:D233-238 [PMID: 18838391].
Enzymatic degradation of yeast cells and yeast cell walls is being investigated already for a long time. Lytic enzymes are produced by bacteria, mycobacteria, streptomycetes and moulds. Several commercially available enzyme products (often containing a mixture of enzyme activities) are widely used for yeast genetics, to fuse or transform yeast cells and to generate protoplasts. The enzyme product Zymolyase® from Amsbio and purified from culture liquid of Arthrobacter luteus, contains a β-1,3-glucan-laminaripentaohydrolase as essential activity. Glucanex® from Novozymes is another product used to make yeast protoplasts, as well as Lyticase from Sigma. All three products contain other activities in addition to a glucanase such as protease and mannanase activity. In more detail these products do not only perform endo-hydrolysis of glucan but also exo-hydrolysis resulting in complete breakdown of the glucan into oligosaccharides and glucose.
A laminaripentaose-producing glucanase (LPHase) is a β-1,3-glucanase that liberates laminaripentaose as the major product from polysaccharides such as laminarin, pachyman or curdlan. Vrsanská et al (1977, Zeitschrift für Allgemeine Mikrobiologie, 17 (6), 465-480) have shown that a laminaripentaose-producing glucanase (glucanase I) from Arthrobacter GJM-1 is one of the enzymes of the yeast lytic system. Incubation of isolated yeast cell walls with glucanase I results in complete solubilisation of the yeast cell walls with the formation of only laminaripentaose.
In JP6192589 (1986—Dainippon Ink & Chemicals), a laminaripentaose-producing-β-1,3-glucanase (LPHase) of Streptomyces matensis DIC-108 is disclosed. The enzyme is used in a process to produce laminaripentaose from the polysaccharides curdlan, pachyman and/or laminarin. The production of the glucanase of Streptomyces matensis DIC-108 is disclosed in JP8173153. JP9262090 and Nakabayashi et al (1998—J.Ferm.Bioeng. 85(5), 459-464)) disclose the cloning and sequencing of the gene encoding the glucanase of Streptomyces matensis DIC-108. The authors also derived the amino acid sequence from the gene. The LPHase of Streptomyces matensis DIC-108 appears to be a unique enzyme since it shows only some amino acid sequence similarity (˜60%) with two other β-1,3-glucanases from Arthrobacter sp.YCWD3 and Oerskovia xanthineolytica respectively which were 99% identical to each other on protein level (Shen et al. (1991) J. Biol. Chem 266(2) pp. 1058-1063 and references cited therein). The crystal structure of the LPHase was solved at 1.62 Å resolution and it turned out that the LPHase belongs to the glycoside hydrolase family 64 (Wu et al. J. Biol. Chem. 284 (39), 26708-26715 “Structure, Mechanistic Action, and Essential Residues of a GH-64 Enzyme, Laminaripentaose-producing β-1,3-Glucanase”). In a recent study, Shresta et al determined the essential amino acids in the enzyme for catalysis (Protein Engineering, Design & Selection vol. 24 no. 8 pp. 617-625, 2011—Characterization and identification of essential residues of the glycoside hydrolase family 64 laminaripentaose-producing-β-1,3-glucanase).
Intact β-glucans (as part of dietary fibre) have interesting health properties, such as stimulation of the immune system in mammals. β-glucans are not digested by mammalian enzymes when orally administered and are taken up in the small intestine where it stimulates mucosal and system immunity by activating both innate and adaptive immunities. First major reported function of β-glucans was antitumor activity. Later reported biological activities include antifungal, anti-infection, radioprotective, cholesterol reduction and postprandial glucose metabolic activities.
The potency of the biological activities of water-soluble and particulate β-glucans is controversial, however it is recently reported that particulate β-glucans have stronger immunostimulating activities than water soluble β-glucans. Further, complete degradation of the glucans of yeast cell walls, either by enzyme mixtures yielding glucose and oligosaccharides or by a laminaripentaose-producing glucanase yielding laminaripentaose, has the disadvantage of the loss of the health properties.
The present inventors have found that intact β-glucans still present in the yeast cell wall matrix have only a limited bioavailability to the mammals when digested in the gastro-intestinal tract. It is therefore a long felt need to, on the one hand increase the bioavailability of the β-glucans in the yeast cell wall and, on the other hand to preserve as much as possible the health properties such as the immune system stimulation. The present invention provides a process whereby β-glucans in yeast cell walls are only partially degraded by a laminaripentaose-producing-β-1,3-glucanase thus increasing on the one hand the bioavailability of the β-glucans and preserving on the other hand health properties such as the immune system stimulation.